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Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis.

Lara-Gonzalez S, Estrella P, Portillo C, Cruces ME, Jimenez-Sandoval P, Fattori J, Migliorini-Figueira AC, Lopez-Hidalgo M, Diaz-Quezada C, Lopez-Castillo M, Trasviña-Arenas CH, Sanchez-Sandoval E, Gómez-Puyou A, Ortega-Lopez J, Arroyo R, Benítez-Cardoza CG, Brieba LG - PLoS ONE (2015)

Bottom Line: In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer.Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model.The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

View Article: PubMed Central - PubMed

Affiliation: IPICYT, División de Biología Molecular, Camino a la Presa San José 2055, CP 78216, San Luis Potosí, San Luis Potosí, México.

ABSTRACT
The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

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GdnHCl-induced denaturation profiles of TvTIM1 and mutants at residue 45, as measured by intrinsic fluorescence emission.(A) I45G, (B) I45A, (C) I45V, (D) I45L, (E) I45F, (F) I45Y and (G) Wild-type TvTIM1. Protein concentration was 10 (black squares), 30 (red circles) or 50 (blue triangles) μg mL-1 in 50 mM Tris-HCl pH 7.4 and 10 mM NaCl, at 25°C. Samples were excited at 280 nm and the data have been normalized for ease of comparison.
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pone.0141747.g004: GdnHCl-induced denaturation profiles of TvTIM1 and mutants at residue 45, as measured by intrinsic fluorescence emission.(A) I45G, (B) I45A, (C) I45V, (D) I45L, (E) I45F, (F) I45Y and (G) Wild-type TvTIM1. Protein concentration was 10 (black squares), 30 (red circles) or 50 (blue triangles) μg mL-1 in 50 mM Tris-HCl pH 7.4 and 10 mM NaCl, at 25°C. Samples were excited at 280 nm and the data have been normalized for ease of comparison.

Mentions: Unfolding studies indicate that TIMs monomers are barely stable [3, 32]. In contrast monomeric TvTIMs are populated as single species facilitating their chemical unfolding characterization. The GdnHCl-induced denaturation curves of monomeric mutants are observed in Fig 4 panels A, B, E and F.


Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis.

Lara-Gonzalez S, Estrella P, Portillo C, Cruces ME, Jimenez-Sandoval P, Fattori J, Migliorini-Figueira AC, Lopez-Hidalgo M, Diaz-Quezada C, Lopez-Castillo M, Trasviña-Arenas CH, Sanchez-Sandoval E, Gómez-Puyou A, Ortega-Lopez J, Arroyo R, Benítez-Cardoza CG, Brieba LG - PLoS ONE (2015)

GdnHCl-induced denaturation profiles of TvTIM1 and mutants at residue 45, as measured by intrinsic fluorescence emission.(A) I45G, (B) I45A, (C) I45V, (D) I45L, (E) I45F, (F) I45Y and (G) Wild-type TvTIM1. Protein concentration was 10 (black squares), 30 (red circles) or 50 (blue triangles) μg mL-1 in 50 mM Tris-HCl pH 7.4 and 10 mM NaCl, at 25°C. Samples were excited at 280 nm and the data have been normalized for ease of comparison.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664265&req=5

pone.0141747.g004: GdnHCl-induced denaturation profiles of TvTIM1 and mutants at residue 45, as measured by intrinsic fluorescence emission.(A) I45G, (B) I45A, (C) I45V, (D) I45L, (E) I45F, (F) I45Y and (G) Wild-type TvTIM1. Protein concentration was 10 (black squares), 30 (red circles) or 50 (blue triangles) μg mL-1 in 50 mM Tris-HCl pH 7.4 and 10 mM NaCl, at 25°C. Samples were excited at 280 nm and the data have been normalized for ease of comparison.
Mentions: Unfolding studies indicate that TIMs monomers are barely stable [3, 32]. In contrast monomeric TvTIMs are populated as single species facilitating their chemical unfolding characterization. The GdnHCl-induced denaturation curves of monomeric mutants are observed in Fig 4 panels A, B, E and F.

Bottom Line: In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer.Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model.The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

View Article: PubMed Central - PubMed

Affiliation: IPICYT, División de Biología Molecular, Camino a la Presa San José 2055, CP 78216, San Luis Potosí, San Luis Potosí, México.

ABSTRACT
The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

Show MeSH
Related in: MedlinePlus